The Reference Gene Selection And Gene Cloning Expression Analysis Of EGF And IF In Onchidium Struma

Onchidium struma was amphibious creature which living in tidal flats wetlands such as brackish water,and belongs to the Mollusca,Gastropoda,Pulmonata,Systellommatophora,Onchidiidae,it has very high edible and medicinal value.The distribution area is widely in Jiangsu,Shanghai,Zhejiang,Fujian,Guangdong and other places of China coast where freshwater and marine salt water were intersection,its ability to adapt to temperature change is very strong,and wild resource is very abundant.The current academic to the O.struma research work mainly concentrated in the system taxonomy,biological characteristics,nutritional value evaluation,fertilization embryo development mechanism and separation of active substances extractin etc.Epidermal growth factor can promote the development of epithelial cell proliferation,Intermediate filament protein play a greatly role in maintaining cell morphology,reference gene screening is a basic step in molecular quantitative experiments.Onchidium struma molting phenomenon involves a large number epithelial cells update,and sports ability while relate to some degree of deformation and movement cells.This paper introduced the reference genes screening for O.struma as well as the functional genes of EGF and IF physical and chemical structure and evolution,and could provide accumulated data for further study on related functional genes of amphibious biology characteristics of O.struma.1.Experiments based on Onchidium struma transcriptome analysis database,select seven sequence which were often used for reference gene as a candidate gene,after the preliminary appraisal on the NCBI BLAST soft,design the fluorescent quantitative PCR primers,and statistical analysis of the stability of candidate genes expression in the different tissue.BestKeeper software analysis showed that the stability: EF1a>TUBB>RPL28>Ubiq>18SrRNA=CYC>ACTB;Similarly,in geNorm software analysis results: EF1a>RPL28>CYC>Ubiq>TUBB>18SrRNA>ACTB;And the steady value of Normfinder software just gives sort: EF1a>RPL28>Ubiq>CYC>TUBB>18SrRNA>ACTB.The comprehensive analysis been known,as the most commonly use referennce genes ACTB standard deviation(SD)is the biggest one,means it stability of gene expression is the lowest in seven candidate genes,and the stability of the highest candidate genes was EF1 a.So ultimately selected EF1 a as reference gene in different tissue expression analysis of the O.struma,while the optimal reference genes combination is EF1 a and RPL28 provide by Normfinder.2.Cloning experiment got the epidermal growth factor(Os-egf1)gene from the dorsal skin of Onchidium struma,and has carried on biological information analysis of the gene sequences,amino acid sequence structure and gene expression.Through use RACE cloning technology,gained the cDNA sequence of Os-egf1 gene is 1158 bp,which open reading frame(ORF)length of 846 bp,and encoding 281 amino acid residues.No obvious signal peptide in the protein sequence,and predict its molecular weight is 30.51 kDa,the gene has an across membrane structure domain(17-39aa).Subcellular localization found the peptide should in the nucleus,theoretical isoelectric point(PI)is 5.41,a partial acidic protein.There are 6 Ser,3 Thr,3 Tyr can be phosphorylation sites for protein kinase.Among the sequence amino acids of Os-egf1 cysteine(Cys)residues is significantly,accounting for 11.40%,mainly distributed in the corresponding three EGF-like structure.These structure field for at least six cysteine residues involved in forming CX7 CX10 CX4-5-13 CXCX8 similar structure,speculated that the three pairs of disulfide bond are significant necessary to maintain specific spatial structure.Remove the cysteine residues in structure domain,the gene sequence of conservative shows very low.The evolutionary tree is displayed that different members of the EGF family get together.Onchidium struma different tissue relative quantitative experiment indicated the Os-egf1 high express in the dorsal skin,due to the epidermal growth factor can promote the proliferation differentiation of epithelial cells,presumed to Os-egf1 gene is associated with the O.struma molting phenomenon.3.Cloning experiment got the Intermediate filament protein(Os-IF1)gene from the dorsal skin of Onchidium struma,and has carried on biological information analysis of the sequences genes,the amino acid sequence structure and gene expression.Os-IF1 gene cDNA sequence length is 1957 bp,the open reading frame length of 1359 bp,and encoding a 452 amino acid residues.No obvious signal peptide in the protein sequence,and predict its molecular weight is 51.54 kDa,the gene has no across membrane structure domain.Subcellular localization found the peptide should in the nucleus,theoretical isoelectric point(PI)is 5.21,a partial acidic protein.There are 23 Ser,14 Thr,6 Tyr can be phosphorylation sites for protein kinase.The Os-IF1 amino acid sequence is made up of primarily ? helix,occupy 82% of the polypeptide chain,which conform to the typical characteristics of intermediate filament protein.The amino acid sequence homology comparison found it appear high similar with the family of intermediate filament protein.A high gene expression level of Os-IF1 expressed in Onchidium struma different tissues is the former tentacles and pleopod,while the other three species the highest organization of expressed is pleopod,presumably the gene product related to abdomen muscle contraction movement process.And Os-IF1 expression analysis among four different species found that the O.struma is the highest in the pleopod,specific reason remains to be further experiments.the Os-IF1 protein sequences conservative is very high,and the evolutionary tree basic performance that O.struma evolutionary relationships closely with Biomphalaria glabrata,and gastropoda,Cephalopoda,bivalve,chordate and higher vertebrates come together form a branch.